This invention is directed to the separation of polynucleotide fragments by liquid chromatography. More specifically, the invention is directed to a system and method, which enhances the chromatographic separation of polynucleotides on non-polar, wide pore separation media.
Separation of polynucleotide mixtures is a focus of scientific interest, and numerous researchers have been attempting to achieve technical improvements in various aspects of polynucleotide separation. Anion exchange separation and reverse phase ion pair chromatography are among the most frequently used methods for separating polynucleotide mixtures.
Samples containing mixtures of polynucleotides can result from total synthesis of polynucleotides, cleavage of DNA with restriction endonucleases or RNA, as well as polynucleotide samples which have been multiplied or amplified using polymerase chain reaction (PCR) techniques or other amplifying techniques.
Previous work has focused on developing rapid, high resolution separations, developing separations based on the size of the polynucleotide fragment rather than the base sequence of the fragment, and on developing the ability to collect separated pure fractions of polynucleotides.
W. Bloch (European patent publication No. EP 0 507 591 A2) demonstrated that, to a certain extent, length-relevant separation of polynucleotide fragments was possible on nonporous anion exchanger separation media using eluting solvents containing tetramethylammonium chloride (TMAC). Y. Ohimya et al. (Anal. Biochem., 189:126-130 (1990)) disclosed a method for separating polynucleotide fragments on anion exchange material carrying trimethylammonium groups. Anion exchangers with diethylaminoethyl groups were used by Y. Kato et al. to separate polynucleotide fragments (J. Chromatogr., 478:264 (1989)).
U.S. Pat. No. 5,585,236 (1996) to Bonn et al. describes a method for separating polynucleotides using what was characterized as reverse phase ion pair chromatography (RPIPC) utilizing columns filled with non-polar, nonporous polymeric beads. High resolution, rapid separations were achieved using an ion pairing agent (triethylammonium acetate), and acetonitrile/water eluting solvent gradient. This work is important because it is the first example of a size dependent, sequence independent chromatographic separation of double-stranded polynucleotides by Matched Ion Polynucleotide Chromatography (MIPC). Such separations are comparable to those effected by gel electrophoresis, which is currently the technology most widely used for polynucleotide separations. Bonn""s work makes it possible to automate separations of polynucleotides based on their size alone. This method differs from traditional reverse phase processes. Therefore, the term Matched Ion Polynucleotide Chromatography (MIPC) has been applied to the Bonn process to distinguish it from previously known reverse phase processes.
The invention of patent application Ser. No. 08/748,376 is based on the discovery that trace levels of multivalent metal ions, even when present below the limits of detection, interfere with the MIPC separation process. Special steps to prevent, remove or complex any trace multivalent ions result in enhanced separation of polynucleotides and lower the detection threshold. The inventions of provisional applications Serial No. 60/049,123 filed Jun. 10, 1997; and Serial No. 60/063,835 filed Oct. 30, 1997 under 35 U.S.C. xc2xa7111(b) are based on the discovery that nitric acid passivated stainless steel, titanium, and PEEK (polyetherether ketone) surfaces were, contrary to popular belief, sources of multivalent metal ion contamination in the MIPC process. The deleterious effect of multivalent metal cations on polynucleotide separations as observed herein has not been previously reported. We believe that all chromatographic processes which are capable of separating polynucleotides on non-polar, wide pore separation media are impaired by the interference of multivalent metal ions.
Therefore, the invention provides an improved method for separating a mixture of polynucleotide fragments wherein multivalent cations are eliminated from the all aspects of the separation process. The method comprises applying a solution of said fragments and counterion agent to a column containing separation media having a non-polar surface, wherein said separation media have a pore size greater than 30 Angstroms and an average diameter of 1-100 microns. Separation of said fragments is accomplished by eluting said fragments with an eluting solvent gradient of increasing organic component concentration containing a counterion agent. Surfaces which are contacted by the solution of the fragments and the eluting solvent are materials which do not release multivalent metal cations therefrom, said materials having been washed to remove traces of organic contaminants therefrom. The method further comprises contacting the solution of said fragments and the eluting solvent with a multivalent cation capture resin to remove any multivalent cations therein before entering the column.
In a preferred embodiment of the invention, the separation media have been treated to remove residual traces of multivalent cations from the surfaces therefrom.
An optimum embodiment of the invention comprises contacting the solution of said fragments and eluting solvent with a multivalent cation capture resin before entering the column, treating the separation media to remove residual traces of multivalent cations from the surfaces therefrom, and ensuring that surfaces which are contacted by the solution of the fragments and the eluting solvent are materials which do not release multivalent metal cations therefrom and cleaning said surfaces to remove any traces of organic contaminants therefrom.
In one embodiment, the polynucleotide fragments are double stranded, having more than 5 base pairs. Such fragments are separated by size or by polarity.
In another embodiment of the invention, the polynucleotide fragments are single stranded having 2 or more nucleotides. Such fragments are separated by size and by polarity.
The separation media are organic polymer, or an inorganic substrate selected from the group consisting of inorganic substrates, silica, zirconia, and alumina. The inorganic substrates support a non-polar material on their surface. Said non-polar material may be organic polymer or long chain, C1 to C24 hydrocarbon groups bound to the inorganic substrate, wherein residual polar groups of the substrate are end capped with trimethylsilyl chloride or hexamethyldisilazane.
In a preferred embodiment, surfaces which are contacted by the solution of polynucleotide fragments and eluting solvent are titanium, coated stainless steel, organic polymer or combinations thereof. Removal of traces of residual multivalent metal cations from the separation process is further ensured by treating said surfaces with a solution comprising aqueous acid and chelating agent, by adding a chelating agent to the solution of polynucleotide mixture and eluting solvent, and by treating the eluting solvent to remove oxygen therefrom.
In one embodiment, the improved method for separating said mixture of polynucleotides comprises Matched Ion Polynucleotide Chromatography.
The improved method of the invention may also be practiced as a batch process for separating polynucleotide fragments having a selected size from a mixture of polynucleotide fragments including fragments of said selected size. The batch process method of the invention comprises applying a solution of said polynucleotide fragments and a counterion agent to non-polar separation media having a non-polar surface, wherein said separation media have a pore size greater than 30 Angstroms and an average diameter of 1-100 microns. The method further comprises contacting the separation media with a first eluting solvent and counterion agent, the first eluting solvent having a concentration of organic component sufficient to release from the separation media all polynucleotide fragments having a size smaller than the selected size and removing the first eluting solvent from the separation media. The selected size fragments are obtained by contacting the separation media with a second eluting solvent having a concentration of organic component sufficient to release from the separation media the polynucleotide fragments having the selected size and removing the second eluting solvent from the separation media. Preferably, surfaces which are contacted by the solution of polynucleotide fragments and the eluting solvent are material which does not release multivalent metal cations therefrom.
Following removal of the first eluting solvent, the separation media are rinsed with fresh first eluting solvent to remove residual released polynucleotide fragments therefrom. In a similar manner, following removal of the second eluting solvent the separation media are rinsed with fresh second eluting solvent to remove residual released polynucleotide fragments of selected size therefrom.
A preferred embodiment of the invention comprises contacting the solution of polynucleotide mixture and eluting solvent with a multivalent cation capture resin before contacting the separation media. In another preferred embodiment the method comprises treating the separation media to remove residual traces of multivalent cations therefrom. Optimally the separation media have been treated to remove residual traces of multivalent cations therefrom and the solution of polynucleotide mixture and eluting solvent have been contacted with a multivalent cation capture resin before contacting the separation media. Said separation media are contained in a column, a web, a membrane, or container.
The batch process can be used to separate mixtures of double stranded polynucleotides or single stranded polynucleotides.
The separation media are organic polymer, or an inorganic substrate selected from the group consisting of inorganic substrates, silica, zirconia, and alumina. The inorganic substrates support a non-polar material on their surface. Said non-polar material may be organic polymer or long chain, C1 to C24 hydrocarbon groups bound to the inorganic substrate, wherein residual polar groups of the substrate are end capped with trimethylsilyl chloride or hexamethyldisilazane.
The surfaces contacted by the solution of polynucleotide fragments and eluting solvent are, preferably, comprised of material selected from the group consisting of titanium, coated stainless steel, and organic polymer, or combinations thereof. Removal of traces of residual multivalent metal cations from the separation process is further ensured by treating said surfaces with a solution comprising aqueous acid and chelating agent, by adding a chelating agent to the solution of polynucleotide mixture and eluting solvent, and by treating the eluting solvent to remove oxygen therefrom.